The incidence of primary and secondary syphilis has nearly doubled in the United States since 2000, and has increased dramatically in Western Europe and China during that time. In developing countries every year at least half a million babies are born with congenital syphilis, and as many cases of stillbirth and miscarriage are caused by this disease. The improvement of our understanding of the pathogenesis of syphilis is a key element to identify new potential vaccine candidates against the disease, but such progress is greatly limited by the current assumption that the agent of syphilis, Treponema pallidum subsp. pallidum (T. pallidum), cannot be genetically manipulated, due to the fact that this pathogen cannot be propagated in vitro. In vitro propagation in fact is regarded as an obligate step for genetic manipulation of a pathogen because it easily provides billions of exponentially growing bacteria able to withstand the elevated mortality of DNA transfer protocols. Most importantly, in vitro propagation allows selection of recombinant strains. Comparative genomics suggests however that, similarly to other pathogens, T. pallidum might be naturally competent for transformation, namely the ability to express enzymes for the intake of exogenous genetic material and its integration into the genome via homologous recombination. Natural competence is widely present among prokaryotes as an effective means to increase genetic diversity but, for T. pallidum it could as well represent an alternative way to obtain genetically altered T. pallidum strains without the need of in vitro propagation. In this application I therefore propose to investigate if T. pallidum is naturally competent for transformation and if this feature can be used to induce targeted integration of a selection marker into the T. pallidum genome. If successful, these experiments will provide an innovative protocol to alter T. pallidum's genome, perform selective gene ablation, and greatly advance our understanding of the function and location of putative virulence factors in relation to the establishment of the infection. PUBLIC HEALTH RELEVANCE: These studies will challenge the assumption that the agent of syphilis, Treponema pallidum (T. pallidum) is impossible to genetically manipulate. Comparative genomics suggests in fact that this pathogen might be naturally competent for transformation, namely able to intake outside DNA and integrate it into its genome if it is similar enough to the bacterium's own DNA. This application will investigate T. pallidum's natural competence and the possibility to use it to develop a genetic modification protocol that will facilitate the identification of new targets for developing effective vaccines to prevent syphilis.